1. Field of the Invention
The present invention relates generally to C-terminal alpha-aminoboronic acid derivatives of lysine, ornithine, and arginine, homoarginine and corresponding isothiouronium analogs thereof, and their use as inhibitors of trypsin-like serine proteases such as thrombin, plasma kallikrein and plasmin.
2. Background
The activity of many biological systems is mediated by hydrolytic or proteolytic enzymes that cleave precurser proteins at specific locations. Four classes of these enzymes exist, metallo, thiol, acid and serine proteases. Systems such as blood coagulation, fibrinolysis, complement, and kallikrein-kinin are all regulated by a subclass of serine proteases, the trypsin-like proteases, a group of enzymes that have a primary specificity for arginyl or lysyl residues.
Within each class, the mechanism of action and the active site residues of the enzymes as well as their susceptibility to class specific inhibitors are similar. The ability of a compound to effectively inhibit a particular protease or a particular subclass of proteases, however, is strongly dependent upon the structure and composition of the compound.
A great deal of research has been done in the area of protease inhibition, and a number of researchers in this area have experimented with boron-containing inhibitors.
Shenvi, U.S. Pat. No. 4,537,773 (1985), for example, reports that alpha-aminoboronic acid analogs of amino acids containing aliphatic and aromatic alkyl side chains are effective inhibitors of metalloenzymes In addition, Shenvi et al., U.S. Pat. No. 4,499,082 (1985) disclose that alpha-aminoboronic acids incorporated into peptides inhibit serine proteases whose primary specificity requirements are met by neutral side chains, such as pancreatic and leukocyte elastase, chymotrypsin, and cathepsin G. This latter patent discloses tetrapeptides comprising C-terminal alpha-aminoboronic acid residues as potent, reversible inhibitors of such proteolytic enzymes. The peptides disclosed, however, did not include C-terminal alpha-aminoboronic acid residues of lysine, ornithine, arginine, homoarginine or any corresponding isothiouronium salts.
Koehler et al., Biochemistry 10: 2477 (1971) report that 2-phenyl-ethaneboronic acid is an inhibitor of chymotrypsin. Matteson et al., J. Am. Chem. Soc. 103: 5241 (1981), describe the synthesis of (R)-1-acetamido-2-phenylethane boronic acid and its use as an inhibitor of chymotrypsin. The authors show a K.sub.i of 4 .mu.M.
Lienhard in Enzyme Inhibitors as Drugs, Sandler, ed., University Park Press, Baltimore pp.43-51 (1980) speculates that peptide analogs of alpha-aminoboronic acids will be potent inhibitors of serine and thiol proteases.
Additional disclosures include those of Kinder et al., J. Med. Chem. 28: 1917-1925 (1985), which describes the N-acyl and dipeptide boronic acids and difluoroborane analogs of phenylalanine, phenylglycine, alanine, valine, and isoleucine, and Matteson, Organometallics 3: 1284-1288 (1984) which describes the synthesis of alpha-amido gama-substituted boronic esters. The latter authors state that these compounds were prepared as possible precursors to boronic acid analogs of arginine and proline.
Trypsin-like proteases are extremely important in controlling a number of physiological processes. For a discussion of such activity, see "Proteases and Biological Control", Reich, Rifkin and Shaw eds., Cold Spring Harbor Press (1975). Thrombin, one type of trypsin-like protease, has a clear and decisive role in the blood coagulation process. Blood coagulation may occur through either of two cascades of zymogen activations. The last protease in each of these pathways is thrombin, which acts to hydrolyze fibrinogen to form fibrin, which in turn aggregates to form a blood clot. This thrombin catalyzed hydrolysis is essential to the blood coagulation process.
Plasma kallikrein, another trypsin-like protease, is also involved in the blood coagulation process, specifically in the initiation of one of the blood coagulation pathways. Also, kallikrein acts on kininogen to liberate the nonapeptide, bradykinin. Bradykinin is a hypotensive peptide that is associated with pain. In addition, kallikrein is thought to have other biological functions. Recent information suggests that plasma kallikrein is involved in inflammation. Baumgarten et al., J. Immun. 137: 977-982 (1986), for example, report elevated levels of kinin and kallikrein in allergic individuals challenged with allergen. Wachtfogel et al., Blood 67: 1731-1737 (1986) report that plasma kallikrein aggregates human neutrophils and releases neutrophil elastase. The release of elastase and accompanying elastase-mediated tissue destruction are events associated with the process of inflammation.
The design of specific inhibitors of trypsin-like enzymes to control biological processes is not a new concept. Particular efforts have been made in the preparation of inhibitors of thrombin to replace heparin in treatment of thrombosis without the side effects associated with heparin therapy, see Markwardt TIPS 153-157 (1980) and Green et al., Thromb Res. 37: 145-153 (1985). Highly effective peptide chloromethyl ketones have been prepared for a number of trypsin-like proteases by Kettner et al., Methods in Enzymology 80: 826-842) (1981). One example, H-(D)Phe-Pro-ArgCH.sub.2 Cl, is highly effective in the inhibition of thrombin (K.sub.i =37 nM), and, as shown by Shaw et al., U.S. Pat. No. 4,318,904 (1982), is effective in the prevention of coronary thrombosis in a rabbit model. Similarly, Bajusz et al., Int. J. Peptide Protein Res. 12: 217-221 (1979) report the peptide aldehyde, H-(D)Phe-Pro-Arg-H, is an effective inhibitor of thrombin (K.sub.i =75 nM) and Tremoli et al., Thromb. Res. 23: 549-553 (1981), report that a related compound, Boc-(D)Phe-Pro-Arg-H, reduces the size of venous thrombosis in rats.
Substituted arginine amides composed of secondary amines have also been shown to be effective inhibitors of thrombin. Kikumoto et al., Biochemistry 23: 85-90 (1984) report that (2R,4R)-4-methyl-1[N.sup.2 -{(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)sulfonyl}-L-arginyl]-2-piperid inecarboxylic acid is an inhibitor of thrombin (K.sub.i =19 nM). As reported by Green et al., Thromb. Res. 37: 145-153 (1985), this inhibitor increases the prothrombin times of plasma in vitro blood coagulation assays 2-fold at 1 .mu.M, and it is claimed as a fibrinolytic enhancing agent to be used in combination with tissue plasminogen activator Yoshikuni et al, European Patent Application 0,181,267 (1986). Finally, Sturzebecher et al., Thromb. Res. 29: 635-642 (1983) and Kaiser et al., Thromb. Res. 43: 613-620 (1986) report that N-alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenyl-alanine piperidide is the most effective known inhibitor of thrombin (K.sub.i =6 nM), and demonstrate that is in vivo efficacy in mice and rats.
Despite the foregoing, new and better classes of inhibitors of thrombin and other trypsin-like enzymes are needed to provide potentially valuable therapeutic agents for treatment of blood coagulation disorders, inflammation and other mammalian ailments. The present invention is directed to this end.